Clinical and chest computed tomography features associated with severe Chlamydia psittaci pneumonia diagnosed by metagenomic next-generation sequencing: A multicenter, retrospective, observational study

Chlamydia psittaci pneumonia is a rare disease with varying clinical presentations. Here, we aimed to investigate the clinical and chest computed tomography (CT) features of severe psittacosis pneumonia. Clinical data of 35 patients diagnosed with psittacosis pneumonia were retrospectively analyzed using metagenomic next-generation sequencing. The patients were classified into severe (n = 20) and non-severe (n = 15) groups. The median age of patients was 54 years, and 27 patients (77.1%) had a definite history of bird contact. Severe patients had more underlying comorbidities and were more prone to dyspnea and consciousness disorders than non-severe patients. The neutrophil count and D-dimer, lactate dehydrogenase, interleukin (IL)-2, IL-6, and IL-10 levels were higher, whereas the lymphocyte, CD3 + T cell, and CD4 + T cell counts, CD4+/CD8 + T cell ratio, and albumin level were substantially lower in severe patients than in non-severe patients. Chest CT findings of severe patients revealed large areas of pulmonary consolidation, and ground-glass opacities were observed in some patients, with a higher risk of involving multiple lobes of the lungs and pleural effusion. One patient died of multiple organ failure, whereas the condition of the other 34 patients improved, and they were discharged from the hospital. Patients with severe psittacosis pneumonia often have underlying comorbidities and are prone to developing dyspnea, consciousness disorder, and lesions in both lungs. Serum D-dimer, IL-2, IL-6, and IL-10 levels and lymphocyte, CD3 + T cell, and CD4 + T cell counts are associated with disease severity.


Introduction
Chlamydia psittaci belongs to the genus Chlamydia, which comprises gram-negative obligate intracellular bacteria including Chlamydia pneumoniae and Chlamydia trachomatis. C psittaci infection often results in severe pneumonia and even multiple organ failure owing to its strong pathogenicity. [1] Pneumonia caused by C psittaci is a rare disease, accounting for approximately 1% of community-acquired pneumonia cases. [2] The number of reported cases is relatively small, mainly because of the limitations of etiological assessment techniques and insufficient clinical information on the disease. [1,3] Medicine of this study was to investigate the clinical and chest computed tomography (CT) features of severe psittacosis pneumonia. For this purpose, we retrospectively analyzed the clinical data of 35 patients with psittacosis pneumonia admitted to 4 tertiary A hospitals in South China between January 2020 and April 2022 and compared the clinical features of severe and non-severe patients.

Study design and participants
In this study, we retrospectively analyzed 35 patients diagnosed with psittacosis pneumonia using mNGS between January 2020 and April 2022 and admitted to 4 tertiary A hospitals in South China. The patients were classified into the following 2 groups based on the severity of pneumonia: severe (n = 20) and non-severe (n = 15) groups. The diagnostic and clinical classification of psittacosis pneumonia was based on the Consensus Guidelines on the Management of Community-Acquired Pneumonia in Adults, developed by the Infectious Diseases Society of America and the American Thoracic Society in 2007; severe pneumonia was defined as the presence of either one major criterion or 3 or more minor criteria. [6] C psittaci DNA was detected in the alveolar lavage fluid or venous blood of all patients using mNGS, and conventional etiological assessments including blood, sputum, and alveolar lavage fluid smears and cultures did not detect other pathogens. Patients aged <18 years, who were pregnant, with thrombotic diseases or HIV infection, and/or with severe data loss, were excluded.

Ethics
This study was approved by the Ethics Review Committee of Huizhou First People's Hospital and was conducted in accordance with the provisions of the Declaration of Helsinki (Approval 2020107). All patients and legally authorized representatives or next of kin of the deceased patients provided informed consent for this study.

Data collection
General information of all patients, including sex, age, underlying diseases, and epidemiological history, was collected. The clinical symptoms and signs of the patients, blood test results, chest CT findings, mNGS results, treatment, and outcome (survival or death) were recorded. The modified British Thoracic Society's pneumonia score (CURB-65), [7] pneumonia severity index (PSI), [8] and acute physiology and chronic health evaluation II [9] score upon hospital admission were calculated.

Detection of pathogens using metagenomic nextgeneration sequencing
The alveolar lavage fluid and venous blood samples were collected in strict accordance with standard clinical operation procedures, and the Ion AmpliSeq Kit (Guangzhou DARUI Biotechnology Co., Ltd., Guangzhou, China) was used for DNA extraction. DNA was randomly cut into 200 to 300-bp fragments. The insert size and adapter sequence were controlled to form a single-stranded ring structure via ligation using the Daan Universal DNA Library Construction Kit (Guangzhou DARUI Biotechnology Co., Ltd.). The prepared DNA nanospheres were loaded onto the sequencing chip and subjected to sequencing and bioinformatic analysis using the DA8600 high-throughput sequencer (Daan Gene Co., Ltd., Guangzhou, China).

Statistical analysis
All statistical analyses were performed using SPSS version 19.0 (IBM Corp, Armonk, NY). Quantitative variables are described as mean (standard deviation) or median (quartiles). A t test was used for intergroup comparisons if the variables were normally distributed, and Mann-Whitney U test was used if the variables were not normally distributed. Categorical variables are described as frequency rate and percentage, and the χ 2 test or Fisher's exact test was used for comparisons between groups. Statistical significance was set at P < .05.

Demographic and clinical characteristics
Of the 35 patients, 16 were males and 19 were females. The median age was 54 years with a range of 30 to 88 years, and 27 patients (77.1%) had a definite history of bird contact. Disease onset was mostly in winter (57.1%, 20 cases). The severe group had higher CURB-65, PSI, and acute physiology and chronic health evaluation II scores and more underlying diseases, and were more prone to dyspnea and consciousness disorders than did the non-severe group ( Table 1, P < .05).
We detected C psittaci DNA in the alveolar lavage fluid (30 cases) and venous blood (5 cases) samples of patients using mNGS. Among them, 11 patients presented this pathogen alone, and the pathogen was more common in non-severe cases. Other pathogenic microorganisms such as Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Candida albicans, and Epstein-Barr virus were also detected in the remaining 24 patients, and were more common in severe cases. The number of reads of C psittaci ranged from 3 to 14 940.

Laboratory and radiological data
The neutrophil count and D-dimer, lactate dehydrogenase, interleukin (IL)-2, IL-6, and IL-10 levels were significantly higher (P < .05), whereas the lymphocyte, CD3 + T cell, and CD4 + T cell counts, CD4 + /CD8 + T cell ratio, and albumin level were significantly lower (P < .05) in the severe group than in the non-severe group ( Table 2).
All patients underwent chest CT after admission (Table 3), and the median time from the onset of the disease to the first chest CT was 5 days. The chest CT showed more frequent and severe exudation in the lower lobes. Consolidation was observed in all patients, and some patients presented ground-glass opacities and a small amount of pleural effusion, pericardial effusion, and mediastinal lymphadenopathy. The lesions in non-severe patients were generally confined to a single lung lobe (Fig. 1). The lesions often progressed rapidly in severe patients, resulting in a massive consolidation of multiple lobes in the lungs and pleural effusion (Fig. 2). After effective treatment, the lesions could be completely absorbed, or only a small amount of strip shadows remained (Figs. 1 and 2).

Treatment and prognosis
All patients underwent mNGS examination after admission, and the median time from admission to diagnosis was 3 days (Table 1). Twenty-seven patients required respiratory support: 10 patients (3 severe and 7 non-severe patients) received oxygen through nasal catheters, 7 severe patients received highflow nasal oxygen therapy, and 10 severe patients required mechanical ventilation with 4 patients receiving tracheal intubation and ventilation and 6 receiving noninvasive ventilation. All patients were treated with antibiotics before diagnosis. The common regimen was β-lactam/β-lactamase inhibitor combinations or carbapenem combined with quinolone. Antiviral www.md-journal.com Table 1 Baseline characteristics of patients with Chlamydia psittaci pneumonia (severe and non-severe groups).  drugs, including oseltamivir and arbidol, were administered to 13 patients, and 8 patients were treated with glycopeptide antibiotics. Of the 15 non-severe patients, 10 were initially treated with respiratory quinolones. After the detection of C psittaci DNA using mNGS, 6 patients continued to use levofloxacin/moxifloxacin, 2 replaced levofloxacin with moxifloxacin owing to a poor response to treatment, and the remaining 7 were treated with doxycycline alone or in combination with other antibiotics. Twenty severe patients were initially treated with respiratory quinolones, of which only 6 showed a good response to treatment, and azithromycin was administered to one of the remaining patients. Tetracyclines were administered alone or with other antibiotics to 13 patients. Except for 1 patient who died of multiple organ failure, all patients showed improvements (Table 4), with their body temperatures gradually decreasing to normal levels in approximately 2 to 3 days.

Discussion
Psittacosis pneumonia is a zoonotic disease caused by C psittaci.
Humans generally lack immunity against the pathogen, and birds are the predominant hosts. Outbreaks caused by person-to-person transmission have been reported, but they are extremely rare. C psittaci is transmitted after the inhalation of aerosolized bacterial cells, [10,11] which can rapidly cause severe respiratory failure and even death. [5,12] High-risk factors for infection include old age, smoking, male sex, glucocorticoid use, and long-term residence in nursing homes. The prognosis of patients is directly associated with immunity. Timely and Table 3 Chest computed tomography manifestations of patients with Chlamydia psittaci pneumonia (severe and non-severe groups).  . The lesions increased, and the scope expanded following treatment with levofloxacin for 7 days (E). Thereafter, the lesion evidently shrank and became less dense following treatment with moxifloxacin for 7 days (F). www.md-journal.com accurate diagnosis and treatment generally lead to a good prognosis. [13,14] The present study showed that most patients had a history of bird contact, and the infection was more common in women and middle-aged people. Most patients had severe pneumonia and often had underlying comorbidities. However, the tracheal intubation rate and mortality rate recorded here were substantially lower than those reported by Tulzo. [15] The possible reasons could be early diagnosis using mNGS, appropriate treatment, and the initial empirical use of quinolones, which improved the condition of some patients. In the past, the CURB-65 and PSI scores were mainly used to evaluate bacterial pneumonia. [16] This study showed that the CURB-65 and PSI scores of patients with severe psittacosis pneumonia were significantly higher than those of non-severe patients, suggesting that the above-mentioned scores performed well in the evaluation of chlamydial pneumonia. C psittaci is a rare pathogen and an intracellular bacterium. When its DNA is detected in a sample using mNGS, the sample is considered positive for the bacterium. [17] In this study, the effect of conventional anti-infection treatment in patients was not good before diagnosis. However,  Table 4 Treatment and outcomes of patients with Chlamydia psittaci pneumonia (severe and non-severe groups). most patients had a good prognosis after the diagnosis of C psittaci infection was established and specific anti-infection treatment was administered. Psittacosis is essentially a systemic infection. After the inhalation of the pathogen through the respiratory tract, it first proliferates in the reticuloendothelial system of the liver and spleen, and then enters the body through blood. Generally, the lungs are primarily affected, and the liver, kidney, and central nervous system could also be affected. [1,18,19] Therefore, the clinical symptoms are diverse with varying severity levels. Common symptoms occurring in the patients evaluated in this study include high fever, cough, dyspnea, fatigue, anorexia, consciousness disorders, headache, and myalgia. Laboratory examinations revealed decreased lymphocyte counts and increased D-dimer, transaminase, lactate dehydrogenase, and creatine kinase levels, indicating that besides the respiratory system disease, the digestive system, neuromuscular system, hematological system, and coagulation function were affected to certain degrees. The more severe the disease, the more acute the organ dysfunction, and this is consistent with the reports of Chen et al [20] and Su et al [21] However, similar to the study of Branley et al, [22] in the present study, only a small number of patients developed renal insufficiency, which might be owing to the small number of patients and timely and accurate diagnosis and treatment that prevented the occurrence and development of a cytokine storm.

Variable
Previous studies have paid little attention to the changes in coagulation function and inflammatory cytokines in psittacosis. In this study, the levels of D-dimer, IL-2, IL-6, and IL-10 in severe patients were remarkably higher, whereas the counts of CD3 + T cells, CD4 + T cells, and lymphocytes were substantially lower than those in non-severe patients. Increased levels of D-dimer indicate a hypercoagulable state and the possibility of vascular endothelial cell impairment. [23] IL-10 is an anti-inflammatory cytokine, whereas IL-2 and IL-6 are pro-inflammatory cytokines. IL-6, which plays a critical regulatory role in the inflammatory response, is a key inflammatory mediator. [24] Currently, the mechanism of a severe Chlamydia infection is not completely clear. Studies have shown that after the activation of the innate immune system by Chlamydia infection, it can promote the release of numerous pro-inflammatory mediators such as IL-6, IL-8, and tumor necrotic factor-α, inhibiting Chlamydia replication and accelerating pathogen elimination. The release of a large amount of pro-inflammatory factors can cause an excessive inflammatory response and coagulation dysfunction in patients. On the contrary, it can activate Treg, Th2, and other cells to secrete IL-10, IL-4, and other immunosuppressive cytokines, leading to a gradual increase in the anti-inflammatory response. A persistent inflammatory response can lead to increased lymphocyte apoptosis and immunosuppression. [24][25][26][27][28] In summary, we believe that severe psittacosis pneumonia induces an excessive inflammatory response and immunosuppression in the early stage, and the lymphocyte counts and D-dimer, IL-2, IL-6, and IL-10 levels can help identify the severity of the disease.
The primary pathological basis for the chest radiologic features of psittacosis pneumonia is inflammatory cell infiltration in the alveolus with fibrinous exudate. [29] The chest X-ray showed different degrees of exudation and consolidation, mainly involving the lower lobes of the lungs, and a small amount of pleural effusion can be observed in some patients. [22,30] Most of the cases reported by Branley et al [22] and Wen et al [29] had single lung lesions, mostly located in the right lung. The chest CT findings of 48 patients reported by Shen et al [31] mostly presented consolidation in a single lung and a single lobe, with a small amount of pleural effusion in a few patients. None of the above studies had analyzed the chest radiologic features of mild and severe patients separately. The chest radiologic findings of the patients in this study were mainly characterized using the degree of lung consolidation. The lesions of non-severe patients were generally confined to a single lung lobe, whereas severe patients mostly had consolidations in multiple lobes of both lungs with a small amount of pleural effusion, suggesting that the degree of consolidation and pleural effusion were closely related to the severity of the disease.
The first-choice treatment for psittacosis pneumonia is doxycycline, and other effective drugs include minocycline, azithromycin, moxifloxacin, and levofloxacin. [11,13] In vitro experiments have confirmed the strong antimicrobial activity of the abovementioned drugs against Chlamydia. [32,33] However, relevant randomized controlled clinical studies are still lacking. Previous studies on psittacosis pneumonia have reported inconsistent results owing to the small number of patients and lack of separate analysis of drug treatment for mild and severe patients. [5,15,29,31] In this study, most patients received quinolones before diagnosis, of which some non-severe patients showed a good response to treatment, whereas severe patients showed a poor response. Therefore, using quinolones alone is not recommended in severe patients. Most patients received tetracyclines alone or in combination with other antibiotics and showed considerable improvement after diagnosis. Chen et al [20] and Wu et al [34] reported good efficacies of tetracyclines against severe psittacosis pneumonia. Therefore, tetracyclines should be the first choice for the treatment of psittacosis. If there are contraindications such as allergy, pregnancy, or use in children, macrolides can be chosen as an alternative treatment. [22] However, considering the high-level resistance of Chlamydia to macrolides, [35,36] only 1 patient was treated with macrolides in this study, and their clinical efficacy needs further verification.
A limitation of the study is that it was a retrospective study with a relatively small number of patients. As the conditions for polymerase chain reaction and serological detection of C psittaci were unavailable, all cases were confirmed using mNGS. Therefore, an in-depth, prospective study with a large number of patients is needed in the future.

Conclusion
In conclusion, patients with psittacosis pneumonia usually have a history of bird contact, and the common symptoms include high fever, cough, anorexia, fatigue, headache, and myalgia. Underlying comorbidities are common in severe patients, who are more prone to dyspnea, consciousness disorders, and multilobe lesions in both lungs than non-severe patients. The levels of serum D-dimer, IL-2, IL-6, and IL-10 and the counts of lymphocytes, CD3 + T cells, and CD4 + T cells could predict the severity of C psittaci pneumonia. Thus, mNGS examination is helpful in early diagnosis, and prompt treatment generally leads to a good prognosis.